The development of new drugs and drug forms has been recently more and more focused on the utilization of polymeric substances, especially water-soluble polymers, as drug carriers. A number of polymeric conjugates of cancerostatic drugs and soluble polymers, in which the tumor drug was attached to the polymer by a non-cleavable covalent bond, a hydrolytically unstable ionic bond or a covalent bond susceptible to enzymatic or simple hydrolysis, have been prepared and studied. This endeavor was aimed to prepare drugs with enhanced pharmacokinetic and pharmacodynamic behavior, enabling targeted therapy of tumors. Polymeric drugs prepared on the basis of HPMA copolymers form an important group. The anti-cancer drug in such substances is attached to a polymeric N-(2-hydroxypropyl)methacrylamide carrier by an enzyme-cleavable oligopeptide sequence, prepared as a substrate for lysosome enzymes (enzymes present in mammalian cells). The structure, synthesis and properties of such conjugates have been described in a patent [Duncan 1985]. The results obtained in this area up to now have been clearly summarized by Kope{hacek over (c)}ek et al [Kope{hacek over (c)}ek et al 2000]. The above polymeric drugs have been effective in the treatment of a number of tumors in mice and rats. At present, two of the polymeric conjugates are even being tested clinically. [Vasey 1999 et al, Julyan et al 1999, Thomson et al 1999]. The results of such clinical testing have shown that a polymeric conjugate of doxorubicin has lower non-specific toxicity than a free drug. Its maximum allowed amount (MAA) is 320 mg/m2 which is 4 to 5 times higher than the clinical amount of free doxorubicin (60-80 mg/m2) normally used. No significant effect on the cardiac functions has been observed after the administration of the polymeric drug, although the individual cumulative amount reached up to 1680 mg/m2. All the other areas of toxicity observed in connection with the administration of free, i.e. non-directed anthracycline antibiotics have been significantly reduced. One of the disadvantages of the polymeric conjugate drugs clinically tested is a relatively low specificity of effect since such conjugates either contain no targeting unit at all, such as PK1, or contain a carbohydrate of a relatively low specificity (galactosamine in the PK2 conjugate in which the ability to direct a polymeric drug to the liver is being tested). Therefore, conjugates in which the aimed specific effect is achieved by attaching a specific targeting molecule (e.g. antibodies, as well as lectin, the growth hormone, transferin, etc.) to the carrier molecule are being developed.
Another disadvantage of the clinically tested conjugates, including those of poly(HPMA) and doxorubicin, is the fact that the drug in its pharmacologically active form is only released from such conjugates in cells by an enzymatic reaction which occurs in lysosomes. This means that the drug is only effective in cells with a high concentration of lysosome enzymes—peptidases. Another disadvantage is a relatively complex structure of the conjugate requiring the inclusion of a sequence from which the drug is released by peptidases, mostly the GlyPheLeuGly (SEQ ID NO:1) tetrapeptide link, which makes the synthesis more expensive and complicated.
Papers provide plenty of information on the preparation and studies of properties of polymers to which a cancerostatic drug is attached by a bond susceptible to hydrolysis in an aqueous medium. The results have been summarized by Kratz [Kratz et al, 1999]. Natural macromolecules, such as albumin, dextrans, transferin, alginates or antibodies, have mostly been used as carriers for cancerostatic drugs. Synthetic polymeric carriers, poly(ethylene glycol) and polyglutamines, have been used in a few cases. The drugs were attached to the carriers by bonds enabling the hydrolysis-controlled release of the active drug both in the extracellular space and inside the cells. In case of doxorubicin (Dox), the bond was most frequently formed by esters of cis-aconityl acid or it was a hydrazone bond. However, in vivo testing of all the above pH-sensitive conjugates in animals has not provided conclusive results. Therefore, none of the polymeric conjugates has been used or clinically tested for tumor treatment.
The polymeric cancerostatic drugs developed by us and prepared on the basis of copolymers of HPMA and a cancerostatic drug linked by a pH-sensitive hydrazone bond have shown, in both in vitro and in vivo tests in mice, a significantly higher anti-tumor efficacy in relation to a number of tumor lines compared to conjugates with the drug attached to the polymeric carrier by an enzyme-cleavable bond via an oligopeptide link. The synthesis of such conjugates compared with the poly(HPMA) conjugates developed earlier is more simple, less expensive and easier to manage since one amino acid only can be used as a link instead of the enzyme-degradable oligopeptide sequence and bonding of the drug to the polymer is a simple reaction. The drug is released as a result of a change of pH in the medium and therefore the presence of lysosome enzymes is not essential for activation. The rate of such release (and therefore the immediate cytostatic concentration) is much higher compared to conjugates containing sequences only degradable by enzymes. [{hacek over (R)}í hová et al, 2001, Etrych et al, 2001].